Gentamicin-thallous-carbonate medium for isolation of fecal streptococci from foods.

Gentamicin-thallous-carbonate (GTC) agar was formulated by Donnelly and Hartman (Appl. Environ. Microbiol. 35:576-581, 1978) to select for fecal streptococci in sewage and water samples. The present study was conducted to determine the usefulness of GTC agar for the enumeration of fecal streptococci in foods. Comparisons were made with KF streptococcal (KF), Pfizer selective enterococcus (PSE), and thallous acetate (TA) agars. Samples of ground beef pork sausage, frozen broccoli, frozen fish, and ice cream were examined. Presumptive streptococcal counts obtained on GTC agar were significantly higher than those obtained on KF and PSE agars and were comparable to those obtained on TA agar. GTC was more sensitive than KF or PSE agars primarily because of the recovery of greater numbers of Streptococcus bovis and Streptococcus equinus strains. Percentages of confirmed fecal streptococci obtained on GTC, KF, PSE, and TA agars were 70, 95, 80, and 74, respectively. Differences between these percentages were not statistically significant, but they indicated that selectivity of GTC agar could be improved. Advantages of using GTC agar to isolate fecal streptococci from foods include a short incubation time (16 to 18 h) and large, distinct colonies that facilitate rapid enumeration and subsequent confirmation.     

Gentamicin-thallous-carbonate medium for isolation of fecal streptococci from foods

Gentamicin-thallous-carbonate (GTC) agar was formulated by Donnelly and Hartman (Appl. Environ. Microbiol. 35:576-581, 1978) to select for fecal streptococci in sewage and water samples. The present study was conducted to determine the usefulness of GTC agar for the enumeration of fecal streptococci in foods. Comparisons were made with KF streptococcal (KF), Pfizer selective enterococcus (PSE), and thallous acetate (TA) agars. Samples of ground beef pork sausage, frozen broccoli, frozen fish, and ice cream were examined. Presumptive streptococcal counts obtained on GTC agar were significantly higher than those obtained on KF and PSE agars and were comparable to those obtained on TA agar. GTC was more sensitive than KF or PSE agars primarily because of the recovery of greater numbers of Streptococcus bovis and Streptococcus equinus strains. Percentages of confirmed fecal streptococci obtained on GTC, KF, PSE, and TA agars were 70, 95, 80, and 74, respectively. Differences between these percentages were not statistically significant, but they indicated that selectivity of GTC agar could be improved. Advantages of using GTC agar to isolate fecal streptococci from foods include a short incubation time (16 to 18 h) and large, distinct colonies that facilitate rapid enumeration and subsequent confirmation.     

Evaluation of Pfizer selective enterococcus and KF media for recovery of fecal streptococci from water by membrane filtration.

Pfizer selective enterococcus (PSE) and KF agars were compared for their recovery of fecal streptococci from sewage effluent on membrane filters. The results showed that PSE agar is highly selective for the enterococci. The tan color resulting from esculin hydrolysis, which was not always visible on the surfaces of the colonies, is not considered a necessary differential characteristic on PSE agar since more than 90% of all colonies recovered on membrane filters were confirmed as fecal streptococci and 86% were confirmed as enterococci. The detection of esculin hydrolysis on membrane filters was not improved by using the new Millipore type HC filter. KF agar recovered significantly greater numbers of organisms but was not as selective, with 83% of the typical colonies being confirmed as fecal streptococci and 54% as enterococci. An attempt to improve the selectivity of KF agar while retaining its inclusiveness by incubation at 45 C was not successful.     

Comparative study of selective media for enumeration of Pseudomonas aeruginosa from water by membrane filtration

In the present study, mPA-D and mPA-E agar, modifications of mPA-C agar that reduce background fecal streptococci that interfere with the differentiation and enumeration of the Pseudomonas aeruginosa colonies grown in other mPA media, are proposed for use in analyzing natural water samples. In addition, the efficiencies of several culture media for the recovery of P. aeruginosa in water after membrane filtration and multiple-tube techniques are compared. The degree of selectivity, precision, efficiency, and sensitivity achieved with the proposed media exceeded that achieved by current methods. Furthermore, they yielded equal rates of accuracy and specificity. Incubation at 36 degrees C resulted in an improved recovery of stressed P. aeruginosa. In conclusion, we propose the use of mPA-D and mPA-E agar, both incubated at 36 degrees C for 24 to 48 h, for analyzing river water and seawater, respectively.     

Comparative study of selective media for enumeration of Pseudomonas aeruginosa from water by membrane filtration.

In the present study, mPA-D and mPA-E agar, modifications of mPA-C agar that reduce background fecal streptococci that interfere with the differentiation and enumeration of the Pseudomonas aeruginosa colonies grown in other mPA media, are proposed for use in analyzing natural water samples. In addition, the efficiencies of several culture media for the recovery of P. aeruginosa in water after membrane filtration and multiple-tube techniques are compared. The degree of selectivity, precision, efficiency, and sensitivity achieved with the proposed media exceeded that achieved by current methods. Furthermore, they yielded equal rates of accuracy and specificity. Incubation at 36 degrees C resulted in an improved recovery of stressed P. aeruginosa. In conclusion, we propose the use of mPA-D and mPA-E agar, both incubated at 36 degrees C for 24 to 48 h, for analyzing river water and seawater, respectively.     

Comparison of Selective Media for Isolation of Presumptive Group D Streptococci from Human Feces

Pfizer Selective Enterococcus (PSE) agar, a medium containing bile, sodium azide, and esculin, was evaluated for its sensitivity and selectivity for detection and enumeration of presumptive group D streptococci in human feces. SF broth and SF broth plus agar (1.5%), representing selective media in common use, were studied simultaneously. Presumptive group D streptococci were recovered on PSE agar from the feces of all 25 subjects. No growth was observed in 8% of specimens in SF broth. No gram-negative organisms were recovered in any medium. PSE agar has the advantages of selecting out Streptococcus bovis, earlier appearance of distinctive reactions, and lack of requirement for special incubation temperature.     

Comparative studies of selective media for recovery of Staphylococcus aureus from natural waters

Several selective media currently used for the enumeration of Staphylococcus aureus from different sources were evaluated in order to establish their quantitative recovery, specificity and degree of selectivity, using different types of water samples. The highest selectivity and reliability in the enumeration of Staph. aureus from the samples was obtained on Borrego-Florido-Romero-0 (BFR-0) and KRANEP agars. The method that produced the highest recovery of Staph. aureus was BFR-0 agar with membrane filter and incubation at 36 degrees C for 48-72 h.     

Comparative studies of selective media for recovery of Staphylococcus aureus from natural waters

Several selective media currently used for the enumeration of Staphylococcus aureus from different sources were evaluated in order to establish their quantitative recovery, specificity and degree of selectivity, using different types of water samples. The highest selectivity and reliability in the enumeration of Staph. aureus from the samples was obtained on Borrego-Florido-Romero-0 (BFR-0) and KRANEP agars. The method that produced the highest recovery of Staph. aureus was BFR-0 agar with membrane filter and incubation at 36°C for 48–72 h.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Enumeration of bifidobacteria in gastrointestinal samples from piglets

The population of Bifidobacterium spp. in fecal samples from suckling piglets was investigated, and Beerens, raffinose-bifidobacterium (RB), and modified Wilkins-Chalgren (MW) agar media were evaluated with regard to the enumeration of bifidobacteria in porcine intestinal samples. The results demonstrated that the population of bifidobacteria in the feces of suckling piglets is numerically low, and a phylogenetic analysis of the 16S rRNA gene from bifidobacterial isolates suggested that a possibly new Bifidobacterium species was isolated. Beerens, RB, and MW agar media were not selective for bifidobacteria in the fecal samples. The highest recovery and diversity of bifidobacteria were obtained for MW agar. Nonbifidobacterial isolates from the three agar media were identified and may contribute to the future formulation of improved selective media for the enumeration of bifidobacteria.     

Enumeration of Bifidobacteria in Gastrointestinal Samples from Piglets

The population of Bifidobacterium spp. in fecal samples from suckling piglets was investigated, and Beerens, raffinose-bifidobacterium (RB), and modified Wilkins-Chalgren (MW) agar media were evaluated with regard to the enumeration of bifidobacteria in porcine intestinal samples. The results demonstrated that the population of bifidobacteria in the feces of suckling piglets is numerically low, and a phylogenetic analysis of the 16S rRNA gene from bifidobacterial isolates suggested that a possibly new Bifidobacterium species was isolated. Beerens, RB, and MW agar media were not selective for bifidobacteria in the fecal samples. The highest recovery and diversity of bifidobacteria were obtained for MW agar. Nonbifidobacterial isolates from the three agar media were identified and may contribute to the future formulation of improved selective media for the enumeration of bifidobacteria.     

Evaluation of Recovery Methods to Detect Coliforms in Water

Various recovery methods used to detect coliforms in water were evaluated by applying the membrane filter chamber technique. The membrane filter chambers, containing pure-culture suspensions of Escherichia coli or natural suspensions of raw sewage, were immersed in the stream environment. Samples were withdrawn from the chamber at regular time intervals and enumerated by several detection methods. In general, multiple-tube fermentation techniques gave better recovery than plating or membrane filtration procedures. The least efficient method of recovery resulted when using membrane filtration procedures, especially as the exposure period of the organisms to the stream environment increased. A 2-h enrichment on a rich, nonselective medium before exposure to selective media improved the recovery of fecal coliforms with membrane filtration techniques. Substantially enhanced recoveries of E. coli from pure-culture suspensions and of fecal coliforms from raw-sewage suspensions were observed when compared with recoveries obtained by direct primary exposure to selective media. Such an enrichment period appears to provide a nontoxic environment for the gradual adjustment and repair of injured cells.     

Evaluation of media for monitoring fecal streptococci in seawater

The selectivity of KF streptococcus agar (KF) for monitoring fecal streptococci (FS) in seawater was examined in 234 samples of Mediterranean water and compared with the selectivity of M-Enterococcus agar (M-Ent) for 124 samples and with bile-esculin-azide agar (BEA) for 17 samples. KF was found to be unsuitable for marine water because Vibrio alginolyticus and other gram-negative bacilli indigenous to this environment grew well on it and produced red colonies identical to those of FS. In 26% of samples, some with high counts of red colonies on the membrane filters (MF), there were no streptococci, only gram-negative bacilli and staphylococci, and in an additional 23.1% the streptococci constituted less than 50% of the "typical" red colonies on the MF. V. alginolyticus also produced FS-like colonies on MF incubated on BEA but was not isolated from MF incubated on M-Ent. Although staphylococci grew and produced FS-like colonies on all three media, M-Ent was the most selective since no gram-negative bacilli were isolated from MF incubated on it.     

Evaluation of media for monitoring fecal streptococci in seawater.

The selectivity of KF streptococcus agar (KF) for monitoring fecal streptococci (FS) in seawater was examined in 234 samples of Mediterranean water and compared with the selectivity of M-Enterococcus agar (M-Ent) for 124 samples and with bile-esculin-azide agar (BEA) for 17 samples. KF was found to be unsuitable for marine water because Vibrio alginolyticus and other gram-negative bacilli indigenous to this environment grew well on it and produced red colonies identical to those of FS. In 26% of samples, some with high counts of red colonies on the membrane filters (MF), there were no streptococci, only gram-negative bacilli and staphylococci, and in an additional 23.1% the streptococci constituted less than 50% of the "typical" red colonies on the MF. V. alginolyticus also produced FS-like colonies on MF incubated on BEA but was not isolated from MF incubated on M-Ent. Although staphylococci grew and produced FS-like colonies on all three media, M-Ent was the most selective since no gram-negative bacilli were isolated from MF incubated on it.     

Isolation and identification of fecal bacteria from adult swine.

An examination of the fecal microflora of adult swine was made with regard to the efficiency of several roll tube media in enumeration and recovery of anaerobes, the effects of medium constituents on recovery, and the isolation and identification of the predominant kinds of bacteria. Total number of organisms by microscopic bacterial counts varied among fecal samples from 4.48 X 10(10) to 7.40 X 10(10) bacteria/g (wet weight). Comparison of different nonselective roll tube media indicated that about 30% of the fecal bacteria could be recovered with a rumen fluid (40%, vol/vol) medium (M98-5). Recoveries of 21 and 15%, respectively, were obtained with M10 and rumen fluid-glucose-cellobiose agar (RGCA) media. Rumen fluid, Trypticase, sugars, and CO2 gas phase were important components required for maximum recovery with this medium. Similar high recoveries of anaerobes were also obtained with M98-5 containing swine cecal extract of place in rumen fluid or M10 plus swine cecal extract. Significantly lower recoveries were observed with RCGA, media supplemented with swine fecal extracts, reinforced clostridial medium, brain heart infusion agar, and prereduced blood agar. Ninety percent of the bacteria isolated from roll tube media were gram positive and consisted of facultatively anaerobic streptococci, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. The remainder of the flora (8%) included several other species of anaerobes and Escherichia coli. Rumen fluid (or volatile fatty acids), Trypticase, and yeast extract additions to basal media stimulated the growth of anaerobic strains. Variation in the relative proportions of the predominant fecal microflora was observed. This work indicates that satisfactory enumeration, isolation and cultivation of the predominant microflora in swine feces can be obtained when strict anaerobic culture methods and a rumen fluid medium are used.     

Primary Klebsiella identification with MacConkey-inositol-carbenicillin agar.

MacConkey-inositol-carbenicillin agar has successfully been used as a primary selective medium for Klebsiella enumeration. With pure cultures, nearly 100% recovery of Klebsiella was observed by membrane filtration. With environmental samples using membrane filtration, 95% of typical pink- to red-colored colonies were verified as Klebsiella, as opposed to only 1% of yellow background colonies. Recovery of Klebsiella on MacConkey-inositol-carbenicillin agar was as good or better than on mEndo agar LES (Difco Laboratories). Recovery and percent colony confirmation with MacConkey-inositol-carbenicillin agar were greater than for other proposed Klebsiella selective media.     

Primary Klebsiella identification with MacConkey-inositol-carbenicillin agar.

MacConkey-inositol-carbenicillin agar has successfully been used as a primary selective medium for Klebsiella enumeration. With pure cultures, nearly 100% recovery of Klebsiella was observed by membrane filtration. With environmental samples using membrane filtration, 95% of typical pink- to red-colored colonies were verified as Klebsiella, as opposed to only 1% of yellow background colonies. Recovery of Klebsiella on MacConkey-inositol-carbenicillin agar was as good or better than on mEndo agar LES (Difco Laboratories). Recovery and percent colony confirmation with MacConkey-inositol-carbenicillin agar were greater than for other proposed Klebsiella selective media.     

Quantitative procedure for enumeration of bifidobacteria.

A membrane filter technique has been developed for the enumeration of bifidobacteria in natural aquatic environments. The technique is quantitative, selective, and differential. The medium (YN-6) contains: yeast extract, 2.0 g; agar, 1.5 g; polypeptone peptone, 1.0 g; vitamin-free Casamino Acids, 0.8 g; sodium chloride, 0.32 g; and L-cysteine hydrochloride, 0.003 g; in 100 ml of deionized water. The medium is adjusted to pH 7.0 before autoclaving. Nalidixic acid (80 micrograms/ml), neomycin sulfate (2.5 micrograms/ml), and bromcresol green (300 micrograms/ml) are included as selective and differential agents. After incubation for 48 h at 37 degrees C in an anaerobic environment, Gram-stained smears from green, glistening, smooth entire colonies are examined microscopically for typical bifidobacterial morphology. No significant difference in recoveries was observed when YN-6 was compared with reinforced clostridial agar, using bifidobacteria freshly isolated from feces and raw sewage. Using this technique with aquatic and fecal samples, less than 9% false-positive and 8% false-negative isolates were observed. These results indicated that the medium was able to satisfactorily recover organisms from a variety of situations.     

Improvement of the membrane filter technique for enumeration of enterococci in water

The membrane filter technique with AC agar medium supplemented with 0.04% NaN3 and 0.00015% 2,3,5-triphenyltetrazolium chloride for enumeration of enterococci in water is described. An appropriate volume of a water sample was filtered through the membrane filter. The membrane filter was put on an AC agar plate (designated as the AC.MF technique), which was incubated at 37 C for 18 hr and further at 45 C for 24 hr. By this AC.MF technique, all the colonies grown on the membrane filters were identified as enterococci, and the count of enterococci obtained by the AC.MF technique was similar to that by the AC.MPN technique. The AC.MF technique may be useful for accurate and rapid enumeration of enterococci in water and serve as a simple method for determining the sanitary quality of water.